1. Field of the Invention
The present invention relates to a human monoclonal antibody to Pseudomonas aeruginosa (hereinafter referred to as "P. aeruginosa"), and its production and use. More particularly, it relates to a human monoclonal antibody, which can recognize a partial structure of O-antigen common to A and H serotype strains of P. aeruginosa, and which shows a binding property to P. aeruginosa of various strains of different serotypes, and its use. It also relates to a hybridoma capable of producing the antibody, and a process for the production of the antibody. The human monoclonal antibody of the invention is useful for prevention and treatment of infectious diseases caused by P. aeruginosa. It is also useful for diagnosis of infectious diseases caused by P. aeruginosa.
2. Description of Related Art
Bacteria causing infectious diseases, i.e. prophlogistic bacteria, are varied with development and change of antibiotics as clinically used. As a result, infectious diseases with bacteria originally having only low pathogenicity or virulence may increase. Thus, P. aeruginosa is currently one of the major pathogenic bacteria causing infectious diseases, of which serious symptoms often lead patients to death, particularly when their immunological competence is low due to continuous administration of immunosupressants, suffering from cancer or burn or the like.
Among various preventive or therapeutic methods for bacterial infections, the most prevailing one is chemotherapy by the use of antibiotics or antimicrobial agents. In fact, there have been developed various antibiotics including streptomycin, kanamycin, penicillin, cephalosporin, etc., which are sensitive to almost all Gram-positive bacteria (e.g. Staphylococci) and Gram-negative bacteria (e.g. E. coli) and produce a prominent clinical effect. However, there are known only few medicinal products active against to P. aeruginosa. Even those medicianl products act on P. aeruginosa only bacteriostatically and not bacteriocidally. Thus, they do not clinically exhibit any remarkable therapeutic effect.
The other preventive or therapeutic method is antibody therapy comprising administration of immunoglobulin. This method is often performed in association with chemotherapy and nowdays attracts much attention as a substitute for chemotherapy. A serum of high antibody titer can be obtained by active immunization of animals such as horse or rabbit, and antibody therapy can be made by administration of such serum. In fact, its remarkable therapeutic effect was proved on experimental infections using various animals. It is known from the cases of diphtheria toxin and viper toxin that antibody therapy using sera originated from animals is quite effective even on human beings. However, introduction of a heterogenous protein obtained from animals into a human body may cause such a serious side-effect as anaphylaxis or any other allergic reaction. It is thus highly desired to develop human immunoglobulin having a high antibody titer against bacteria and showing a prominent therapeutic effect on bacterial infections.
Conventional human immunoglobulin preparations are manufactured by collecting blood from healthy persons or bacteria-infected patients, subjecting the blood to fractionation to obtain an immunoglobulin fraction, purifying the immunoglobulin fraction and eliminating agglutinating materials therefrom by addition of ethylene glycol, treatment with protease, sulfonization, DEAE-column chromatography, etc., followed by formulation of the resulting product into intramuscularly or intravenously injectionable preparations. These preparations are advantageous in not causing anaphylaxis or any other side-effect as seen on administration of immunoglobulin originated from animals but they have some drawbacks. One such drawback is that their antibody titer against bacteria is low so that a sufficient therapeutic effect can not necessarily be produced. Another drawback is that their stable supply with a high antibody titer in a large amount is difficult, because they are manufactured using blood collected from healthy persons or bacteria-infected patients and the constant and continuous obtainment of sera having a high antibody titer is quite hard. A further drawback is that they may be contaminated with hepatitis virus (e.g. HB virus), Adult T cell leukaemia virus (ATLV, HTLV), etc., because the blood as the starting material is obtained from a number of unknown persons. In order to overcome these drawbacks, production of a human monoclonal antibody having a strongly protective effect on the infections with P. aeruginosa is highly desirable.
When an antibody is bound to the surface layer of a bacterial body, the phagocytosis of a macrophaze on the bacterial body is accelerated (i.e. acceleration of phagocytosis due to opsonization), or the lysis of the bacterial body by a complement takes place. As the target antigen at the surface layer of the bacterial body of P. aeruginosa, there are known lipopolysaccharide (LPS), outer membrane protein, flagellum, pilus, etc. Of these, Sawada et al. reported that a far large amount of a mouse monoclonal antibody recognizing the outer membrane protein is required in combatting bacteria as compared with a mouse monoclonal antibody which recognizes LPS (J. Infect. Dis., 150, 570-576 (1984)).
LPS consists of O-polysaccharide which represents O-antigen, an outer core oligosaccharide being common among species to some extent, an inner core oligosaccharide, of which component of saccharide, i.e. heptose or 2-keto-3-deoxyoctonate (KDO), is generally almost common to all enterobacteria, and lipid A. The O-polysaccharide antigen locating at the outermost surface of a bacterial cell consists of repeating units of 2 to 5 sugar residues, and its structure varies to a large extent. The structures of almost all O-antigens of the standard serotype strains of P. aeruginosa have already been determined (Eur. J. Biochem., 106, 643-651 (1980); ibid., 125, 229-237 (1986); ibid., 150, 541-550 (1985); ibid., 155, 659-669 (1986); ibid., 167, 549-561 (1987)).
Because determination of the structure of O-antigen by chemical analysis requires a lot of time and labor, antisera or mouse monoclonal antibodies against the O-antigens from the standard strains are employed for the classification of the strains of P. aeruginosa. Namely, an unknown strain of P. aeruginosa is classified in accordance with an immunological reactivity with known antibodies or antisera. This classification is known as a serotype, and typical examples of the serotype classification are as follows: Types 1 to 17 according to the classification by Homma et al. (Japan J. Exp. Med., 44, 1, (1974)); Types 1 to 7 according to the classification by Fisher et al (J. Bacteriol., 98, 835 (1969)); Types A to M according to the classification by Nippon Ryokunoh-kin Kenkyukai Kesseigatabetsu Kento Iinkai (Committee of Study on Serotype Classification, Japanese Study Group on Pseudomonas aeruginosa (hereinafter referred to as "Japanese Committee") (Japan J. Exp. Med., 45, 329 (1976)); Types 1 to 17 according to the classification by International Antigenic Typing System (IATS), etc. These classifications and their cross-relations are shown in Table 1 (Japan J. Exp. Med., 46, 329 (1976)).
TABLE 1 ______________________________________ Serotype classification of P. aeruginosa Japanese Commitee Homma et al ITATS Fisher et al 1976 1974 1983 1969 ______________________________________ A 1 3 -- B 2, 7, 13, 16 2, 5, 16 3, 7 C 3 8 6 D 4 9 -- E 5 11 2 F 6 4 -- G 8 6 1 H 9 10 5 I 10 1 4 J 11 15 -- K 12 13 -- L 14 -- -- M 15, 17 -- -- -- -- 7, 12, 14, 17 -- ______________________________________
It is known that the antibody specific to a certain O-antigen shows a strong preventive or therapeutic effect for P. aeruginosa infections of the serotype to which said O-antigen belongs but does not show any effect against P. aeruginosa in other serotypes. Consequently, for the production of prophylactic and therapeutic preparations by the use of the O-antigen specific monoclonal antibody, it is necessary to broaden its binding spectrum by mixing (hereinafter referred to as "cocktailing") plural monoclonal antibodies, respective antibodies being specific to respective O-antigens in different serotypes. However, the use of plural monoclonal antibodies necessitates the increase of production steps such as cultivation of antibody-producing cell lines, and purification of the monoclonal antibody therefrom makes the entire process very complicated, thereby raising problems in productivity and production cost, etc.
As stated above, the monoclonal antibody against outer membrane protein, like conventional antibodies against LPS core, is not sufficiently effective, although the binding spectra of the former is fairly broad in P. aeruginosa. Therefore, elimination of the problems as seen in production of the cocktail preparation comprising the anti O-antigen monoclonal antibodies is highly desirous. Namely, if one could produce a monoclonal antibody which specifically recognizes an O-antigen and exerts a significant prophylactic and therapeutic effective against infections caused by P. aeruginosa, while exerting a broad binding spectra to a large number of serotype strains, the cocktail preparation comprising said antibody may possibly require least numbers of other monoclonal antibodies. In other words, production of the cocktail preparation usable for a wide range of the serotype strains could be accomplished, even if it comprises least numbers of such monoclonal antibodies.
Fukuda et al. made a report on a specific human monoclonal antibody reactive with several O-antigens of P. aeruginosa (WO88/04669). However, this report only demonstrated that the monoclonal antibody binds to two types of LPS derived from different serotype standard strains of P. aeruginosa, and is still silent on whether said monoclonal antibody can specifically recognize the O-antigen in LPS. Further, on the basis of the reactivity shown with regard to one or two LPS derived from standard strains of P. aeruginosa, it is hardly difficult to conclude that such monoclonal antibody has a specific binding property to other strains within the same serotype. More specifically, Fukuda et al disclosed in their report eleven species of the monoclonal antibody (HPs 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12), each being reactive to the LPS derived from standard strains of P. aerusinosa in Types D and I, E and F, A and L, G and H, E and F, and A and F, but no disclosure was made to the monoclonal antibody capable of specifically recognizing either LPS in Types A and H, particularly O-antigen therein.
Likewise, Zweerink et al. reported that they established a human monoclonal antibody which could bind to LPS of P. aeruginosa in serotypes B and C (corresponding to types 3, 6 and 7 according to Fisher's serotype classification) (Infect., Immun., 56, 1873-1879 (1988); JP-A-63-107999). Similar to the report by Fukuda et al., this report is suggestive of a certain antigen in LPS, which their monoclonal antiobdy specifically recognizes, but so far no specific disclosure was made on that antigen.